I am hoping to perform TEM on exosomes enriched/isolated by ultracentrifugation from cell lines.
In an effort to save time and money during optimization; can anybody suggest a good concentration of exosomes for adsorption?
The plan is to adsorb fixed exosomes onto glow discharged carbon-Formvar coated 200-mesh Cu grids and stain with uranylacetate.
Thank you,
Sam
Hi Samuel,
Once you have your isolated exosome prep I would simply put a droplet onto parafilm and float an EM grid on top of the exosome solution. The time you incubate the grid with the exosomes will most likely define how many end up adhered to the grid so you can try out a few incubation times and see what will work best. Also dilute from your initial exosome prep if you need less vesicles on the grid.
One comment about the contrasting: try out contrasting the vesicles in a mix of methylcellulose and uranylacetate (according to the Tokuyasu method for immunogold labelling), this will give you a much more consistent staining over the entire grid compared to simple negative staining. See link for details: https://www.researchgate.net/file.PostFileLoader.html?id=56eacfa1615e27a907713491&assetKey=AS%3A340651913826304%401458229153561
Hope this helps,
good luck,
Christian
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